Abstract

Ischemic stroke remains a leading cause of global mortality and disability. While timely vascular recanalization is the most direct and clinically validated intervention for cerebral ischemia, reperfusion often induces secondary brain injury, with neuroinflammation playing a central role. Single-cell sequencing data from an ischemic stroke mouse model identify G protein-coupled receptor 35 (GPR35) as a potential regulator of post-ischemic inflammatory responses. GPR35 expression was markedly increased in both in vivo cerebral ischemia–reperfusion models and in vitro oxygen–glucose deprivation systems, predominantly localizing to microglia. Functional studies revealed that genetic knockdown of GPR35 in murine brain tissue significantly exacerbated cerebral infarction volume, neurological deficits, and neuroinflammation in animal models, whereas GPR35 overexpression produced therapeutic effects. Consistently, pharmacological activation of GPR35 using zaprinast attenuated ischemia–reperfusion injury and reduced proinflammatory cytokine production. Mechanistically, zaprinast-mediated GPR35 activation suppressed proinflammatory cytokine production via modulation of the Raf1/ERK1/2/MAPK signaling cascade. Notably, Raf1 knockdown attenuated the pathological exacerbation induced by GPR35 deficiency in peri-infarct regions. Co-immunoprecipitation analyses revealed a direct interaction between GPR35 and Raf1, with the CR2 domain, a critical region for Raf1 autoinhibition, identified as the primary binding interface. Collectively, these findings demonstrate that zaprinast confers cerebroprotective effects in cerebral ischemia–reperfusion injury by activating GPR35, ultimately attenuating infarct progression and neuroinflammation. This mechanistic insight positions GPR35 as a promising therapeutic target for mitigating reperfusion injury in ischemic stroke.